This invention relates to the diagnosis of rheumatoid arthritis, a chronic disease of the joints and other tissue that is a serious health problem worldwide. In particular, the present invention is directed to providing a predictive test for rheumatoid arthritis in its early stages which shows high specificity and sensitivity, enabling prompt and accurate diagnosis and hence effective treatment with appropriate drugs. Such early treatment can limit irreversible joint damage which is known to occur within the first few years or even months after the onset of rheumatoid arthritis.
Rheumatoid arthritis is the most serious of the rheumatic disorders in terms of population prevalence, potential for crippling and morbidity, and life-shortening effects. The disease is characterised by the symmetrical inflammation of multiple joints (polyarthritis). It most frequently affects the small joints of the hands and feet, but inflammation can occur in virtually any joint including spinal joints. Pain, stiffness and swelling of the joints are the main symptomatic features, resulting in loss of function. Damage to the joints leads to serious deformities and functional impairment. Apart from the effects on the joints, rheumatoid arthritis may be associated with a wide range of extra-articular features affecting various organs, such as the heart, blood vessels, lungs and kidneys. Although these extra-articular features are most common in the case of serious forms of rheumatoid arthritis, they may also provide the first symptom of the disease. There is at present no reliable cure for rheumatoid arthritis. Treatment is essentially directed towards alleviating the discomfort caused by the symptoms and arresting the progression of the disease. Sometimes, however, the disease appears to resolve spontaneously, or in response to one or other of the drug regimens currently employed.
Rheumatoid arthritis generally appears after puberty. The prevalence rises with age, and it is 2-3 times more frequent among women than men. The prevalence of definite rheumatoid arthritis is between 1-2% in the majority of white populations (Hochberg, MC, 1981), and the treatment of patients with rheumatoid arthritis consumes a significant component of the health care budget.
Rheumatoid arthritis is included among the autoimmune diseases. Many authors assume that exposure to an infectious agent, bacterium or virus, can initiate rheumatoid arthritis in individuals with a genetic predisposition to the disease. The actual disease is generated by an abnormal reaction of the immune system, which then plays a central role in the progression of articular damage and extra-articular lesions. Since no infectious agent has in fact been convincingly implicated in the disease, rheumatoid arthritis may be a spontaneously occurring autoimmune process, in which the primary response is to an autoantigenic component of the joint itself, rather than to an extrinsic infectious agent.
The idea that rheumatoid arthritis is an autoimmune response to a component of cartilage is traced back to Steffen and Timpl (1963) who first showed antibodies to collagen in rheumatoid arthritis and proposed that rheumatoid arthritis results from an autoimmune response to the collagen molecule present in cartilage now know to be type II collagen. This idea is strongly supported by the observation that immunization with type II collagen induces an arthritis with similarities to human rheumatoid arthritis in appropriate strains of rats, mice or primates (Courtney et al 1980, Trentham et al, 1977).
In human rheumatoid arthritis, autoantibodies to native and denatured type II collagen are detectable in the serum and synovial fluid of up to 30% of patients according to data derived from cross-sectional studies on patients with generally long-standing disease (Morgan et al, 1987; Terato et al, 1990; Rowley et al, 1992). However, the importance of such antibodies to type II collagen has long remained controversial in view of their low frequency in most reported studies, the lack of correlation between antibody levels and disease status (Clague et al, 1980b Stuart et al, 1983; Collier et al, 1984; Morgan et al, 1989; Stockman et al, 1989) and the reported presence of these antibodies in a range of disease other than rheumatoid arthritis (Clague et al, 1980; Trentham et al, 1981; Gioud et al, 1982; Rosenberg et al, 1984; Charriere et al, 1988; Choi et al, 1988; Rowley et al, 1988, 1992). As mentioned, most of the positive associations of about 30% between antibodies and disease have been based on patients with rheumatoid arthritis of long duration. More recently, several studies have shown that the frequency of autoantibodies to type II collagen may be as high as 60-75% in patients tested very early in the course of rheumatoid arthritis, and levels of autoantibody tend to fall as the disease progresses to levels ascertained in the earlier cross-sectional studies (Pereira et al, 1985; Fujii et al, 1992, Cook et al, 1994, 1996). Accordingly, it has been proposed that antibodies to collagen will provide a useful predictive marker of early rheumatoid arthritis and particularly so for those patients in whom rapid progression to joint destruction will occur.
The main structural proteins of the connective tissue in the body are collagens, of which at least 19 genetically different types have so far been described (Brodsky and Shah, 1995). The types of collagen found in a specific tissue are related to the function of the tissue, and they have specific distributions within individual tissues. Articular cartilage in mature joints contains a number of different collagen types, of which type II collagen is the most abundant. It is the major fibrous collagen in all hyaline cartilage and represents 80-90% of the collagen content. Its role is to build up a fibrous network which, together with proteoglycans and hyaluronan, creates an extremely strong structure with the capacity to withstand high pressures (Heinegard and Paulsson, 1987). Type II collagen is a highly conserved molecule between species. It consists of 3 identical a chains, and is moderately glycosylated (Miller, 1985). It is restricted to cartilage and few other tissues, these being the vitreous humour of the eye, and intervertebral discs, in contrast to the more universal distribution of type I and III collagens (Gay et al, 1980).
In general, the basic structure of all native collagen consists of three polypeptide xcex1-chains in the form of a triple helical domain(s) with repeating glycine-X-Y triplets, in which X is often proline and Y is often hydroxyproline (Piez, 1982). Hydroxyproline is essential for the formation of hydrogen bonds that stabilize the helix. Each xcex1-chain is coiled into a tight left-handed helix which averages about 3 amino acid residues per twist. Three xcex1-chains coil about one another in a right-handed manner to create a 300 nm long, 1.5 nm thick, superhelix which is stabilized by hydrogen bonds formed between the xcex1-chains. About 25 to 30 amino acid residues are required on each chain to complete one turn of the superhelix. Heat denaturation of collagen molecules at 45xc2x0 C. leads to unfolding of the triple helix to display the linear sequence of amino acids along the length of the individual xcex1-chains.
Collagen is stabilized by the formation of covalent cross-links. Two kinds of cross-links are formed in the collagen fibre, intra- and inter- molecular. Cross-link formation involves enzymatic conversion of lysine and hydroxylysine residues, to allysine and hydroxyallysine respectively, by peptidyl lysine oxidase. Allysine and hydroxallysine react with each other, or with lysine, spontaneously, to form aldol and aldimine condensation products. No enzyme catalysis is required for this process, only the physical proximity of the appropriate side chain (Miller, 1985). Cross-links are formed between a modified lysyl or hydroxlysyl residue in the telopeptide region and a hydroxylysyl residue in the conserved triple helical region, and the amount of cross-linking increases with the age of the individual.
The intact triple helical domain of collagen is resistant to almost all enzymes except mammalian collagenase which cleaves most collagens into a three-quarter length TcA fragment and a one-quarter length TcB fragment. However, upon heating, the triple helix is denatured and thus susceptible to non-specific proteinases. Cleavage of collagen with cyanogen bromide, which cleaves at the carboxyl terminal of methionine residues, produces a unique set of peptides, called CB peptides, which can be purified by a combination of ion-exchange and molecule sieve chromatography (Piez, 1976). For type I collagen, CB peptides have been numbered using rat collagen as the standard, and for type II collagen, CB peptides have been numbered using chick collagen as the standard. When a methionine cleavage site is missing as may occur in collagen of another species, the numbers designating the 2 uncleaved peptides are both used, separated by a comma (see FIG. 1). Separated xcex1-chains and CB peptides are able to renature, under appropriate conditions, to their original triple-helical conformation (Terato et al, 1985). CB-peptides of type II and type II collagen are particularly amenable to renaturation when separated, because in each type the three xcex1-chains are identical (Terato et al, 1985, Werkmeister and Ramshaw, 1991).
As noted above, the type II collagen molecule consists of three chains wound in a triple helix, and techniques do not yet exist that allow production of a xe2x80x9cnativexe2x80x9d type II collagen molecule by genetic engineering from cDNA constructs. For this reason, measurement of antibodies to collagen requires the use of collagen purified from cartilage, whether of human or animal origin, or purified from chondrosarcoma cell lines. However, since most healthy people have low levels of natural antibody to collagen in their serum, perhaps representing a response to dietary collagens, tests designed to measure antibodies to collagen in rheumatoid arthritis using the entire molecule of purified collagen will not necessarily give disease-specific results. Thus, it is preferable to identify regions of the collagen molecule that react specifically with antibodies in sera of patients with rheumatoid arthritis.
In the work leading to the present invention, the regions of the collagen molecule (epitopes) that are recognized by antibodies to collagen have been examined by Western blotting, using cyanogen bromide digests of collagen, whereby the collagen molecule is cleaved at methionine residues, to produce a number of CB peptides (see FIG. 1). Most studies on antibodies to CB peptides of collagen in rheumatoid arthritis disclose responses to multiple peptides (Terato et al, 1990; Buckee et al, 1990; Rowley et al, 1992; Cook et al, 1994). In the past, however, no significant difference has been found in the pattern of recognition of CB peptides by patients with rheumatoid arthritis or other diseases (Rowley et al, 1992), except that antibodies to type II collagen in the inflammatory disease of cartilage, relapsing polychrondritis, react with one of the smaller CB polypeptides of collagen, CB9,7, which is rarely recognized by patients with rheumatoid arthritis (Terato et al, 1990).
In a first aspect, the present invention provides a method for the detection of antibodies to collagen in a biological sample from a patient, the method comprising contacting said biological sample with an antigen comprising the CB10 peptide of mammalian type II collagen, or an antibody-binding fragment or variant thereof, for a time and under conditions for an antibody-antigen complex to form, and then detecting said complex.
It will, of course, be apparent to persons skilled in this art based on the disclosure herein that the detection of an antibody-antigen complex in accordance with the method of this invention is an indicator of rheumatoid arthritis in the patient, and whilst there is no simple definitive test for rheumatoid arthritis, the method of this invention enables the detection of antibodies to collagen as a predictive marker of early rheumatoid arthritis.
The antigen used in the method of the present invention comprises the CB10 peptide of mammalian type II collagen, or an antibody-binding fragment or variant thereof, from any suitable source, particularly bovine or human type II collagen. The CB10 peptide, or antibody-binding fragment thereof, may be produced by cleavage of the whole collagen molecule with cyanogen bromide, followed by purification of the CB10 fraction, as is well known in the art. Alternatively, however, a similar fraction of the collagen molecule may be prepared by other protein chemistry techniques, including solid phase and other protein synthesis techniques.
The method of the present invention utilises an immunoassay format, including for example an ELISA format, which is well known to persons skilled in this art.
The present invention also extends in another aspect to a diagnostic kit for the detection of antibodies to collagen in a biological sample from a patient, which comprises, in compartmental form, an antigenic preparation comprising the CB10 peptide of mammalian type II collagen, or an antibody-binding fragment or variant thereof, and means for detection of a complex formed between said antigen and antibodies to collagen in said biological sample.
Preferably, the biological sample is a serum, plasma or whole blood sample from the patient. Preferably also, the biological sample is taken from a human patient.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.